Antimalarial acridines: Synthesis, in vitro activity against P. falciparum and interaction with hematin

Article abstract of DOI:10.1016/j.bmc.2009.10.005

A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure-activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC₅₀ ≤ 0.07 μM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC₅₀ ≤ 0.3 μM. These acridine derivatives inhibited the formation of β-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.

Full text of DOI:10.1016/j.bmc.2009.10.005

Bioorganic & Medicinal Chemistry 17 (2009) 8032–8039
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial acridines: Synthesis, in vitro activity against P. falciparum
and interaction with hematin
Lucie Guetzoyan a, , Xiao-Min Yu a, Florence Ramiandrasoa a, Stéphanie Pethe a, Christophe Rogier b,
Bruno Pradines b, Thierry Cresteil c, Martine Perrée-Fauvet a, Jean-Pierre Mahy a,
*
a Équipe de Chimie Bioorganique et Bioinorganique, Institut de Chimie Moléculaire et des Matériaux d’Orsay, Bât. 420, CNRS UMR 8182, Univ Paris-Sud, 91405 Orsay Cedex, France
b Unité de Recherche en Biologie et Epidémiologie Parasitaires, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR 6236,
Institut de Médecine Tropicale du Service des Armées, Bd Charles Livon, Parc Le Pharo, 13998 Marseille Armées, France
c Ciblothèque cellulaire, Institut de Chimie des Substances Naturelles, CNRS, Gif sur Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated
against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and
FCR3) of Plasmodium falciparum. Structure–activity relationship showed that two positives charges as
well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antima-
larial activity. The best compounds possessing these features inhibited the growth of the chloroquine-
Received 15 July 2009
Revised 2 October 2009
Accepted 5 October 2009
Available online 9 October 2009
susceptible strain with an IC50 6 0.07
l
M, close to that of chloroquine itself, and that of the three chlo-
M. These acridine derivatives inhibited
Keywords:
Antimalarial
Plasmodium falciparum
Aminoacridine
b-Hematin
roquine-resistant strains better than chloroquine with IC50 6 0.3
l
the formation of b-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally,
in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-
ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial
activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain
and promising selectivity on other strains.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
used, that involves the synthesis of 6-chloro-2-methoxyacridine
derivatives.5–7 9-Aminoacridines and quinolines (like CQ) are
Malaria is the third cause of death per infectious disease after
tuberculosis and AIDS as it affects 200–500 million people and
causes 1–2 million fatalities every year.1 Quinine, and then chloro-
quine (CQ, Chart 1) were employed in the past five decades to fight
malaria not only because of their ease of use and low cost, but also
because they presented very few side effects. Unfortunately resis-
tant strains of Plasmodium falciparum have appeared, however
resistance mechanisms seem to involve CQ itself rather than a
modification in the drug targets,2 as it is explained in the following
paragraph.
known to be potent DNA intercalators8 and can inhibit DNA tran-
scription in parasites.9–11 However, it is now commonly admitted
N
HN
Chloroquine
(CQ)
Cl
N
+
+
R
1 = OCH3, R2 = Cl
1 = OCH3, R2 = Cl
R1 = OCH3, R2 = Cl
R3 = NH3
R3 = CH3
R3 = Acr
n = 1, 2, 3
n = 3
1-3
4
R3
n
A new class of antimalarial agents, acridines, and especially 9-
aminoacridines (quinacrine and 9-aminoacridines), has been devel-
oped in the last 15 years.3,4 In addition, a particularly promising
strategy for optimising their antimalarial activity has been recently
HN
R
R1
n = 3
5
6
N
R2
R
1 = R2 = H
R3 = NH3
n = 3
O
+
X=
NH3
7
8
Abbreviations: BOP, benzotriazol-1-yloxy-tris(dimethylamino) phosphonium
hexafluorophosphate; CDI, carbonyldiimidazole; CQ, chloroquine; DIEA, diisopro-
pylethylamine; TFA, trifluoroacetic acid.
* Corresponding author. Tel.: +33 1 69 15 74 21; fax: +33 1 69 15 72 81.
E-mail address: jpmahy@icmo.u-psud.fr (J.-P. Mahy).
HN
X
O
X=
+
N
H
NH3
N
Present address: Department of Biological Sciences, University of Warwick,
Gibbet Hill road, Coventry CV4 7AL, UK.
Chart 1. Chloroquine and acridine derivatives tested in this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.005

L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
8033
that the main reason for which they are toxic for the parasite stands
in the fact that they interfere with the crystallization of free haem
generated during the degradation of hemoglobin.12–16 This consti-
tutes a particularly interesting property since the haem detoxifica-
tion pathway does not seem to be dependent on any specific
enzyme, which, consequently, makes the appearance of resistance
harder. Thus, CQ has been extensively used for 20 years before the
emergence of resistance, whereas one year was sufficient for the
parasite to develop resistance against pyrimethamine (inhibitor of
dihydropteroate synthase, an enzyme involved in the metabolism
of folate).17 Two other arguments also lead to consider that the haem
detoxification pathway still represents a valid target for the discov-
ery of new anti-malarial drugs. First, in the haem detoxification
pathway, the ‘drug receptor’ is free haem that doesn’t exist at such
a concentration under physiological conditions in human cells. It
then represents a better target in comparison to some parasite en-
zymes that are analogous to those found in human (various prote-
ases, farnesyl transferase and enzymes involved in choline
uptake). Second, the haem detoxification pathway is not by itself di-
rectly involved in quinoline resistance, whereas drug transporters
(PfCRT) are through their involvement in the alteration of drug accu-
mulation into the parasite which results from a reduced uptake of
the drug, an increased efflux, or a combination of the two pro-
cesses.18 In fact, for most of resistant Pf strains, this transporter gene
(pfcrt) present point mutations leading to acidification of the diges-
tive vacuole and efflux of CQ.
derivatives 1–4, while the bis-acridine derivative 5 was obtained
by modifying the relative proportion of 1,6-diaminohexane
(0.5 equiv with respect to acridine) (Scheme 1).
Surprisingly, addition of an excess of 1,6-diaminohexane to 9-
chloroacridine, yielded the bis-acridine compound as major prod-
uct. Consequently, the protection of one of the amino substituents
of 1,6-diaminohexane was necessary and the acridine derivative 6
was obtained in two steps, using mono tert-butoxycarbonyl-diami-
nohexane, followed by classical TFA treatment to remove the pro-
tecting group (Scheme 2a). Two 9-amido acridine derivatives were
also synthesised and studied (Scheme 2b). The acridine derivative
7 was synthesised with the workup procedure reported earlier
from our laboratory.7 The acridine derivative 8 was prepared by
two successive peptidic coupling reactions using b-alanine units
protected by a tert-butoxycarbonyl group.
Both elemental analysis and spectral data (1H and 13C NMR, UV–
vis, ES MS, HRMS, or microanalysis) of the synthesised compounds
are in agreement with the structures of the products.
3. Antimalarial activity, interaction with hematin and
cytotoxicity
The antimalarial activity was tested on one CQ-susceptible P.
falciparum strain 3D7 (Africa), and three CQ-resistant strains W2
(Indochina), FCR3 (the Gambia) and Bre1 (Brazil). The antimalarial
activity was quantified from the inhibition of parasite growth. Re-
sults are reported as the micromolar inhibitory concentrations nec-
In a previous paper,7 we described the synthesis and the in vitro
antimalarial activity of a series of 9-substituted acridine deriva-
tives. Some of them showed significant antimalarial activities
essary to reduce 3H-hypoxanthine incorporation by 50% (IC50
Table 1).
,
against CQ-resistant strains with IC50 values 60.20 lM. In this pa-
Interactions with the haem detoxification pathway were evalu-
ated by different approaches. b-Hematin crystallization inhibition
assays were performed using a slightly modified procedure of the
b-hematin crystallization inhibitory activity (BHIA) assay, de-
scribed by Parapini et al.24 The experimental conditions used
per, we report the synthesis and the in vitro antimalarial activity of
new molecules containing the same scaffold (i.e., 9-aminoacri-
dine). These compounds are listed in Chart 1. Two novel shortened
chain analogues (compounds 1 and 2) of 9-(6-ammoniohexylami-
no)-6-chloro-2-methoxyacridinium dichloride, the more efficient
formerly studied compound (compound 3) were synthesised. We
expected them to be more potent than CQ against CQ-resistant
strains, as it was already observed in the case of 4-aminoquinoline
analogues of CQ with shortened alkylamino side chains.19,20 A 9-
heptylaminoacridine (compound 4) was also synthesised and its
antimalarial activity was compared to that of compound 3, in order
to evaluate the role of the terminal positive charge of 9-amino-sub-
stituent. One bis-acridine derivative 5, whose anti-malarial activity
was already described in the literature,5 was included in this study.
It will be useful to evaluate the contribution of two protonated
acridine rings. Compound 6 differs from 3 only by the absence of
6-chloro and 2-methoxy substituents on the acridine ring. Finally,
two 9-amido acridine derivatives (compounds 7 and 8) whose
study will be helpful to assess if a positive charge on the acridine
ring is necessary to confer a significant antimalarial activity, were
synthesised. Among all the compounds tested, compounds 4 and 8
are new acridine derivatives never published to our knowledge.
The synthesis of the other ones was already described (1–3,21,22
(hemin, DMSO, pH 5) afford the optimal reaction to detect
p–p
interactions between haem and drug. The acetate buffer maintains
the pH of the reaction in the appropriate range as pH 5 corresponds
to the isoelectric point of hematin and allows the best
p–p com-
plexation between hematin and aromatic compounds like acri-
dines. It is noteworthy that this pH coincides also with the
estimated pH of the parasite food vacuole.25 Results are reported
as the concentration that is necessary to reduce hematin crystalli-
zation by 50% (IC50).
NH
n
3 Cl
n = 1 1
n = 2 2
n = 3 3
HN
O
N
H
Cl
i, ii
Cl
HN
Cl
N
O
H3CO
iii
N
Cl
Cl
6
23), but their antimalarial activities were never evaluated except
4
for the compound 77 and the bis-acridine derivative 5.5 In addition,
experiments were performed to correlate the antimalarial activity
of molecules 1–8 with their ability to interact with hematin and
prevent the crystallization of free haem.
iv
Cl
Cl
N
O
H
N
N
H
2. Chemistry
N
5
O
The 9-amino-6-chloro-2-methoxyacridine derivatives 1–5 were
prepared from commercial 6,9-dichloro-2-methoxyacridine
according to already described procedures.7,22 Addition of an ex-
cess of diaminoalkane afforded the 9-amino substituted acridine
Scheme 1. Synthesis of compounds 1–5. Reagents and conditions: (i) H2N–(CH2)n–
NH2, 80 °C, 4 h; (ii) TFA/CH2Cl2 1:1, rt, 16 h, then DOWEX Clꢀ; (iii) n-heptylamine,
80 °C, 4 h; (iv) 1,6-hexanediamine, DIEA, DMF, 80 °C, 16 h.

8034
L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
O
Cl
N
NH
3 Cl
HN
N
iii, ii
7
i, ii
NH2
N
Cl
NH3
HN
O
O
O
HN
N
NH3 TFA
iv, ii
HN
N
H
NH3 Cl
iii, ii
N
H
Cl
N
6
8
(a)
(b)
Scheme 2. Synthesis of compounds 6–8. Reagents and conditions: (i) H2N–(CH2)n–NHBoc, DIEA, 60–80 °C, 6 h; (ii) TFA/CH2Cl2 1:1, rt, 16 h, then DOWEX Clꢀ (no DOWEX for 8
synthesis); (iii) BOP, DIEA, DMF, rt (50 °C for 8 synthesis), 16 h; (iv) CDI, DMF, rt, 16 h.
Table 1
Comparative in vitro efficiency of chloroquine (CQ) and the acridine derivatives against chloroquine-susceptible and chloroquine-resistant P. falciparum strains
The stoichiometry and the dissociation constant Kd of the hema-
tin/acridine complex, as well as the pKa of the acridine moieties
were also determined, using spectrophotometric methods in both
cases. All these results are presented in Table 2.
In vitro cytotoxicity of the synthesised compounds was also
evaluated upon human KB cells, a cell line derived from a human
carcinoma of the nasopharynx system and expressed as the micro-
molar concentrations (IC50) causing 50% of cell death (Table 3).
magnitude than CQ on CQ-susceptible strain 3D7, as they show
IC50 values in the submicromolar range (42 nM for 1 and 67 nM
for 2). These values are lower than that displayed by 3 on this
strain (130 nM). The antimalarial activity tested on CQ-resistant
strains also shows that three compounds (1–3) are more active
than chloroquine; a fourth compound, 6, is also more efficient than
CQ on FCR3 and Bre1 strains. Replacement of the 6-chloro and 2-
methoxy substituents by an hydrogen atom (acridine derivatives
6 vs 3) decreases the inhibitory potency by threefold on CQ-resis-
tant strains and by ninefold on CQ-susceptible strain 3D7. A larger
decrease of antimalarial activity is observed when the terminal po-
sitive charge on the 9-side chain is removed. Indeed, depending
upon the strains, 4 is 73–151-fold less potent than 3. The antima-
4. Results and discussion
Antimalarial activities are summarised in Table 1. Compounds 1
and 2 display an antiplasmodial potency of the same order of

L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
8035
Table 2
complexes are reported in Table 2. First, The IC50 values were in the
same order of magnitude (0.3–4.6 mM) as the one exhibited by CQ
(0.9 mM), the reference compound, which suggested that this class
of compounds also acted on haem crystallization. Second, the ratio
for the stabilized complex between hematin and most of the acri-
dine derivatives tested under these conditions (2–4 and 6) was
found to be 3:2, which is close to that obtained for CQ (2:1). A
1:1 ratio was obtained with the bis-acridine derivative 5; which
could be explained in this case by the fact that one hematin may
be stacked between the two acridine moieties. The same ratio
was also found for the shortest acridine derivative 1.
Biophysical data: pKa and interaction with hematin
a
Compound
pKa
BHIA
Complex [hematin]/[drug]
IC50 (mM)
Stoichiometry
Kd (lM)
CQ
1
2
3
4
5
6
7
8
8.10
7.94
9.10
9.20
7.50
8.23
9.86
4.59
4.53
0.9
4.6
1.0
0.4
1.5
1.3
0.3
1.1
1.8
2:1
1:1
3:2
3:2
3:2
1:1
3:2
nd
5.4
4.2
8.1
4.4
5.4
20
4.6
nd
4.0
Thecharacterisationof allhematin/drug complexesgiveaccessto
3:2
Kd values, which are all in the same range (from 4.2 to 8.1
lM), ex-
a
pKa values were determined using spectroscopic methods in aqueous medium,
cept for compound 5 (Kd = 20 M). Those discrepancies in Kd and
l
150 mM NaCl. All compounds were dissolved before use either in water or DMSO.
stoichiometry values obtained for the bis-acridine derivative 5
(compared to CQ and the other compounds) are likely due to the sec-
ond acridine moiety, but unexpectedly this second protonated acri-
dine does not improve the affinity for hematin. Similar Kd and
hematin crystallizationIC50 values were obtained when the 6-chloro
and 2-methoxy substituents are replaced by hydrogen (acridine
derivatives 6 vs 3). However, acridine derivative 6 has a lower
anti-malarial activity than 3 (threefold on CQ-resistant strains and
upto ninefoldonCQ-susceptiblestrain3D7, Table1), suggestingthat
in this particular case the antiplasmodial potency could not be en-
tirely explained by the interaction with hematin. Indeed, the proton-
ation state of 6 (pKa = 9.86) may affect its ability to pass through the
membrane of the food vacuole, which could explain the difference of
antimalarial activity, at least on CQ-susceptible strains.
Table 3
In vitro cytotoxicity and selectivity index
Compound
IC50 KB (
l
M)
Selectivity Index (SI)
3D7
W2
FCR3
Bre1
CQ
1
2
3
4
5
6
7
8
22.5a
2.07a
0.60b
0.70b
0.85b
0.05b
0.79a
52.9a
60.1a
1250
50
8.9
5.4
0.04
0.3
0.7
1.3
1.5
51
45
43
7
2
4.1
0.05
0.045
1.8
2.0
2.8
8
6
2.4
3.9
0.02
0.01
1.3
1.1
2.9
2.4
3.5
0.06
0.05
2.2
1.8
3.4
The best inhibition of hematin crystallization is obtained with
compounds that bear one positive charge on the acridine moiety
(whatever the substitution on it), and one positive charge on the side
chain at pH 5, the distance between those two charges being also a
significant factor. Indeed, the highest inhibition of hematin crystal-
lization is found with the compound bearing the hexyl chain
(IC50 = 0.4 and 0.3 mM, respectively, for acridine derivatives 3 and
6), whereas the 6-ammoniobutylamino-acridine derivative 2 and
the bis-acridine compound 5 display a lower inhibition comparable
to CQ (IC50 = 0.9–1.3 mM). A lower inhibition is also observed when
there is only one positive charge (IC50 = 1.5 and 1.8 mM, respec-
tively, for compounds 4 and 8). Strikingly, one of the most potent
compounds on parasite (acridine derivative 1) showed the weakest
hematin crystallization inhibition (IC50 = 4.6 mM). Nevertheless,
acridine derivative 1 displays a pKa value similar to that of CQ, it will
thus be trapped like CQ, once inside acidic compartments (i.e., food
vacuole).28 This behaviour may explain the good antimalarial po-
tency of this compound on the CQ-susceptible strain 3D7.
SI was calculated as the ratio of IC50 for in vitro cytotoxicity on KB to the IC50 of
plasmodial inhibition.
a
IC50 values determined in duplicate.
IC50 values determined in triplicate.
b
larial activity is however partially restored when a second posi-
tively charged acridine is grafted (5). However, as can be seen from
Table 1, the IC50 values measured for bis-acridine 5 are ranging
from 1.01 lM to 4.55 lM, showing a strong variability between
different CQ-resistant Plasmodium strains, and are rather disap-
pointing when compared to the values previously published by
Girault et al.5 Taken altogether, those first results on compounds
1–6 then suggest that both the presence of the chloro and methoxy
substituents on the acridine ring and that of two positive charges
at physiological conditions are required to get a good antimalarial
activity.
An even more important loss of inhibitory potential is observed
when the only positive charge is located on the side chain with a
neutral acridine moiety (compound 7 vs compound 3) with IC50
values 160–320-fold higher in this case. Such a loss of antiplasmo-
dial effect was already observed for CQ analogues, and it was also
shown that chemical modifications that imply the loss of the quin-
oline positive charge beared by the heterocycle of CQ tended to
have a more pronounced impact on antimalarial activity than did
modifications of the side chain.26,27 By contrast to CQ analogues,
acridine compounds display here similar potency against CQ-resis-
tant strains when the only positive charge is located either on the
acridine nucleus (4) or at the end of the side chain (9-amidoacri-
dine 8). Those later results on compounds 7 and 8 then confirm
those obtained with compounds 1–6, and, in summary, the results
of Table 1 clearly indicate that both the presence of 6-chloro and 2-
methoxy substituents on the acridine ring and that of two positive
charges, one on the acridine ring and one on the terminal function
of the side chain, are required for an optimal anti-malarial activity.
The inhibition of hematin crystallization was evaluated for all
the compounds and the IC50 values measured by the BHIA method
as well as the stoichiometry and the Kd value for the drug/hematin
The comparison of the various characteristics of the hematin/
drug complexes described in Table 2 then suggests that the main
interaction between hematin and the various drugs occurs between
the acridine ring and the porphyrin ring system, which could be
hydrophobic as well as electrostatic.29,30 This has been confirmed
by the UV–vis spectra (data not shown) that display significant
hypochromic shifts (ꢀ40%). Indeed, such hypochromicity values
are comparable with those obtained in the case of porphyrin interac-
tions with DNA,31,32 giving evidence for
p–pstacking interactions. In
addition, the optimal chain length grafted on the 9-position appears
to be a hexyl chain which suggests that an electrostatic interaction
may occur between the haem propionate groups and the terminal
ammonium group of the acridine derivatives 3 and 6.
In vitro cytotoxicity was assessed by using KB cells and the
selectivity index (SI) calculated as the ratio of IC50 for in vitro cyto-
toxicity on KB cells to the IC50 of plasmodial inhibition on CQ-sus-
ceptible and -resistant parasites.33,34 The results are presented in
Table 3. Among the most active compounds on P. falciparum, com-
pound 1 exhibits a low toxicity against KB cells line, with a selec-
tivity index ranging from 7 for CQ-resistant strains to 50 for the

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L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
CQ-susceptible strain 3D7. Acridine derivative 2, whose antimalar-
ial activity is similar to 1, displays more toxicity and therefore less
selectivity towards plasmodia, with a selectivity index ranging be-
tween 2 and 9. Selectivity indexes are comparable for the com-
pound 3, whatever the strains; acridine derivatives 6, 7 and 8
show no selectivity at all and compounds 4 and 5 show even more
cytotoxic properties than antimalarial activity.
to an already described procedure22 and obtained in 92% yield.
75 mg of N1-(6-chloro-2-methoxy-acridin-9-yl)-ethane-1,2-dia-
mine was then dissolved in TFA (2 mL) and stirred overnight at
room temperature. TFA was then evaporated and the solid was dis-
solved in distilled water and passed over an ion exchange resin
Dowex Clꢀ. The chloride salt, 9-(6-ammonioethylamino)-6-
chloro-2-methoxyacridinium dichloride 1, was obtained quantita-
tively, after lyophilization.
1H NMR (CD3OD, 250 MHz) d ppm: 2.98 (t, J = 6.50 Hz, 2H–CH2
2), 3.75 (t, J = 6.50 Hz, 2H–CH2 1), 3.93 (s, 3H, O–CH3), 7.20 (dd,
J1 = 9.50 Hz, J2 = 2.16 Hz, 1H–CH Acr 70), 7.33 (dd, J1 = 9.50 Hz,
J2 = 2.88 Hz, 1H–CH Acr 30), 7.38 (d, J = 2.88 Hz, 1H–CH Acr 10),
7.74 (d, J = 9.50 Hz, CH Acr 40), 7.78 (d, J = 2.16 Hz, 1H–CH Acr 40),
8.12 (d, J = 9.50 Hz, CH Acr 80).
5. Conclusion
In summary, convenient, affordable and rapid syntheses were
used to obtain potent antimalarials with aminoacridine as scaffold.
Structure–activity relationship showed that under physiological
conditions two positives charges, one on the acridine ring and
the other at the end of a 9-(6-ammonioalkylamino) chain were re-
quired to exert a good antimalarial activity as well as 6-chloro and
2-methoxy substituents on the acridine ring. Experiments carried
out with hematin suggest that these compounds likely act by inhi-
bition of the haem crystallization process. Indeed, all of the acri-
dine derivatives described here are able to interact with hematin
but different hematin/drug complexes are obtained with structur-
ally close compounds, which is still not fully understood. Besides,
even if all of the efficient compounds are able to inhibit haem crys-
tallization, no tight correlation was found between antimalarial
activity and hematin crystallization IC50 values. Nonetheless, the
acridine derivative 3, the most potent compound on CQ-resistant
strains, is the best inhibitor of haem crystallization.
Finally, among the compounds presented, 1 displayed promis-
ing antimalarial activity in vitro against P. falciparum with quite
good selectivity index versus mammalian cell on the CQ-suscepti-
ble strain (3D7) and promising selectivity on other strains (W2,
FCR3, Bre1) that could be optimised on the basis of further SAR
studies. Future work will focus on this compound which could be
a lead for further structural modifications.
13C NMR (CD3OD, 63 MHz) d ppm: 43.2 (CH2 2), 53.1 (CH2 1),
56.1 (O–CH3), 100.8 (CH Acr), 116.0 (C Acr), 118.6 (C Acr), 124.4
(CH Acr), 126.0 (CH Acr), 126.7 (CH Acr), 126.9 (CH Acr), 130.2
(CH Acr), 136.3 (C Acr), 146.7 (C Acr), 148.9 (C Acr), 152.5 (C
Acr), 157.2 (C Acr).
MS (ES) m/z: 302.1 (MH+) (100%).
HRMS calcd for C16H16ClN3O (MH+): 302.1060, found: 302.1050.
UV–vis (H2O): kmax nm (e
molꢀ1 L cmꢀ1) = 274 (38 900), 349
(4090), 422 (6 540), 442 (7 330).
6.1.2. 9-(6-Ammoniobutylamino)-6-chloro-2-methoxyacridi-
nium dichloride (2)
This compound was synthesised from 6,9-dichloro-2-methoxy-
acridine (1.0 g, 3.59 mmol) and 1,4-butanediamine (20 mL),
according to an already described procedure22 and obtained in
93% yield. 75 mg of N1-(6-chloro-2-methoxy-acridin-9-yl)-bu-
tane-1,2-diamine was then dissolved in TFA (2 mL). The mixture
was stirred overnight at room temperature, then TFA was evapo-
rated. The solid was dissolved in distilled water and passed over
an ion exchange resin Dowex Clꢀ. The chloride salt, 9-(6-ammoni-
obutylamino)-6-chloro-2-methoxyacridinium dichloride 2, was
obtained quantitatively, after lyophilization.
1H NMR (CD3OD, 360 MHz) d ppm: 1.55 (qn, J = 7.40 Hz, 2H–
CH2 3), 1.82 (qn, J = 7.40 Hz, 2H–CH2 2), 2.65 (t, J = 7.40 Hz, 2H–
CH2 4), 3.84 (t, J = 7.40 Hz, 2H–CH2 1), 3.97 (s, 3H, O–CH3), 7.29
(dd, J1 = 9.36 Hz, J2 = 2.16 Hz, 1H–CH Acr 70), 7.42 (dd,
J1 = 9.36 Hz, J2 = 2.88 Hz, 1H–CH Acr 30), 7.53 (d, J = 2.88 Hz, 1H–
CH Acr 10), 7.82 (d, J = 9.36 Hz, CH Acr 40), 7.86 (d, J = 2.16 Hz,
1H–CH Acr 40), 8.27 (d, J = 9.36 Hz, CH Acr 80).
6. Materials and methods
6.1. Chemistry
Most of the reactions were carried out in the dark. All other sol-
vents and reagents were pure grade and used without purification.
The following commercially available chemicals were pure grade
and used as received: 9-aminoacridine, ethylene diamine, 1,
4-diaminobutane and n-heptylamine (Acros), 6,9-dichloro-2-meth-
oxyacridine and 1,6-hexanediamine (Aldrich). Synthesis of 9-(6-
ammoniohexylamino)-6-chloro-2-methoxyacridinium dichloride
3 was reported earlier from our laboratory.7
Reactions were monitored by thin layer chromatography (TLC)
performed on silica gel sheets containing UV fluorescent indicator
(60 F254 Merck). 1H and 13C NMR spectra were recorded on Bruker
AC 200, Bruker AC 250, Bruker AV 360 and Bruker AC 400 spec-
trometers (200, 250 and 400 MHz for 1H, respectively, and 50,
63, 90 and 100 MHz for 13C, respectively). Chemical shifts, d, are re-
ported in ppm taking residual CDCl3 or CD3OD as the reference.
Mass spectra were recorded on a Finningan-MAT-95-S, using
MeOH/CH2Cl2/H2O (45:40:15, v/v) as solvent. High-resolution
mass spectrometry analyses were performed by the ‘Service de
Spectrométrie de Masse de l’ICSN’ Gif-sur-Yvette. Elemental analy-
ses were performed by the Service Central de Microanalyse du
CNRS de Gif-sur-Yvette.
13C NMR (CD3OD, 63 MHz) d ppm: 30.1 (CH2), 29.5 (CH2), 30.5
(CH2), 41.9 (CH2), 56.2 (O–CH3), 101.2 (CH Acr), 114.9 (C Acr),
115.8 (C Acr), 118.4 (C Acr), 124.2 (CH Acr), 126.0 (CH Acr), 127.0
(CH Acr), 127.0 (CH Acr), 130.3 (CH Acr), 136.4 (C Acr), 149.2 (C
Acr), 152.8 (C Acr), 157.2 (C Acr).
MS (ES) m/z: 329.1 (MH+) (100%).
HRMS calcd for C18H20ClN3O (MH+): 330.1373, found: 330.1369.
UV–vis (H2O): kmax nm (
060), 422 (5380), 442 (4760).
e
molꢀ1 L cmꢀ1) = 277 (17 800), 341 (4
6.1.3. N1-(6-Chloro-2-methoxy-acridin-9-yl)-heptylamine (4)
A mixture of 6,9-dichloro-2-methoxyacridine (1 g, 3.59 mmol)
and heptylamine (3.75 equiv, 20 mL) was heated at 80 °C for 4 h.
The mixture was diluted with CH2Cl2, washed with water and dried
over Na2SO4. After filtration and evaporation, the crude product
was purified by chromatography over silica gel with CH2Cl2/CH3OH
(99:1 to 96:4, v/v) to afford N1-(6-chloro-2-methoxy-acridin-9-yl)-
heptylamine 4 in 61% yield (780 mg, 2.18 mmol).
1H NMR (CD3OD, 360 MHz) d ppm: 0.82 (t, J = 6.84 Hz, 3H–CH3
7), 1.20 (m, 4H–CH2 5 + 6), 1.29 (m, 4H–CH2 3 + 4), 1.73 (qn,
J = 6.84 Hz, 2H–CH2 2), 3.73 (t, J = 6.84 Hz, 2H–CH2 1), 3.93 (s,
3H–O–CH3), 7.22 (dd, J1 = 9.36 Hz, J2 = 2.16 Hz, 1H–CH Acr 70),
7.36 (dd, J1 = 9.36 Hz, J2 = 2.88 Hz, 1H–CH Acr 30), 7.44 (d,
6.1.1. 9-(6-Ammonioethylamino)-6-chloro-2-methoxyacridi-
nium dichloride (1)
This compound was synthesised from 6,9-dichloro-2-methoxy-
acridine (1.2 g, 4.30 mmol) and ethanediamine (20 mL), according

L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
8037
J = 2.88 Hz, 1H–CH Acr 10), 7.78 (d, J = 9.36 Hz, CH Acr 40), 7.82 (d,
6.1.6. 7-(Acridin-9-ylcarbamoyl)-heptyl-ammonium chloride
(7)
J = 2.16 Hz, 1H–CH Acr 40), 8.16 (d, J = 9.36 Hz, CH Acr 80).
13C NMR (CD3OD, 90 MHz) d ppm: 14.2 (CH3), 22.9 (CH2), 27.3
(CH2), 29.4 (CH2), 31.7 (CH2), 32.1 (CH2), 55.8 (O–CH3), 100.4 (CH
Acr), 114.8 (C Acr), 117.3 (C Acr), 124.0 (CH Acr), 125.4 (CH Acr),
125.6 (CH Acr), 126.0 (CH Acr), 128.2 (C Acr), 136.1 (C Acr), 145.2
(C Acr), 147.6 (C Acr), 152.5 (C Acr), 156.3 (C Acr).
This compound was synthesised from 9-aminoacridine and NH-
Boc-caprylic acid according to the procedure already described.7
The crude product was then passed over ion exchange resin Dowex
Clꢀ; compound
lyophilisation.
7 was obtained in quantitative yield after
MS (ES) m/z: 357.2 (MH+) (100%).
1H NMR (CD3OD, 250 MHz) d ppm: 1.40 (m, 6H–CH2 3, 4, 5),
1.70 (m, 2H), 1.90 (m, 2H–CH2 6), 2.80 (t, 2H–CH2 1), 2.90 (m,
2H–CH2 7), 7.20 (t, 1H–CH Acr), 7.50 (t, 1H–CH Acr), 7.70 (dd,
2H–2 CH Acr), 8.10 (dd, 2H–2 CH Acr), 8.30 (d, 1H–CH Acr).
13C NMR (CD3OD, 50 MHz) d ppm: 26.3 (CH2), 27.3 (CH2), 28.5
(CH2), 29.9 (CH2), 30.1 (CH2), 37.4 (CH2), 40.7 (CH2), 112.3, 119.5,
120.7, 122.7, 124.9, 125.1, 127.2, 128.5, 136.6, 138.3, 140.2,
141.5, 153.4, 175.5 (CO amide).
Anal. Calcd for C21H25ClN2Oꢁ0.5H2Oꢁ0.2CH2Cl2: C, 66.50; H,
6.95; N, 7.32. Found: C, 66.52; H, 7.03; N, 7.24. MM = 392.5.
UV–vis (DMSO): kmax nm (e
molꢀ1 L cmꢀ1) = 286 (14 500), 345
(1 190), 426 (3 070), 450 (2 500).
6.1.4. N,N1-Bis-(6-chloro-2-methoxy-acridin-9-yl)-hexane-1,6-
diamine (5)
A
solution of 6,9-dichloro-2-methoxyacridine (1.66 mmol,
460 mg), 1,6-hexanediamine (0.83 mmol, 97 mg) and DIEA
(1.66 mmol, 274 L) was heated at 80 °C in DMF for 16 h. After
MS (ES) m/z: 336.3 (MH+) (100%).
HRMS calcd for C21H25N3O (MH+): 333.2076, found: 336.2079.
Anal. Calcd for C21H26ClN3Oꢁ0.5NaClꢁ2H2O: C, 57.70; H, 6.92; N,
9.32. Found: C, 57.71; H, 6.91; N, 9.32. MW: 437.16.
l
concentration under reduced pressure, water was added with stir-
ring. The resulting solid was filtered and purified by MPLC with
CH2Cl2/MeOH (5–15% MeOH) to afford N,N1-bis-(6-chloro-2-meth-
oxy-acridin-9-yl)-hexane-1,6-diamine 5 in 17% yield (780 mg,
2.18 mmol).
UV–vis (H2O): kmax nm (e
molꢀ1 L cmꢀ1) = 251 (130 900), 358
(11 200), 422 (8 540).
6.1.7. Synthesis of2-[2-(acridin-9-ylcarbamoyl)-ethylcarbamoyl]-
ethyl-ammonium chloride (8)
6.1.7.1. [2-(Acridin-9-ylcarbamoyl)-ethyl]-carbamic acid tert-
butyl ester. A mixture of Boc-b-AlaOH (4.22 mmol, 800 mg), BOP
1H NMR (CDCl3, 360 MHz) d ppm: 1.41 (qn, J = 6.8 Hz, 4H–2 CH2
2), 1.69 (t, J = 6.8 Hz, 4H–2 CH2 3), 3.63 (q, J = 6.8 Hz, 4H–2 CH2 1),
3.91 (s, 3H O–CH3), 4.58 (t, J = 6.8 Hz, 2H–2 NH), 7.15 (d, J = 2.5 Hz,
2H–2 CH Acr 50), 7.28 (dd, J1 = 1.8 Hz, J2 = 9.4 Hz, 2H–2 CH Acr 30),
7.40 (dd, J1 = 2.5 Hz, J2 = 9.4 Hz, 2H–2 CH Acr 70), 7.96 (d, J = 9.4 Hz,
2H–2 CH Acr 40), 7.98 (d, J = 9.4 Hz, 2H–2 CH Acr 80), 8.05 (d,
J = 1.8 Hz, 2H–2 CH Acr 10).
(5.07 mmol, 2.24 g) and DIEA (5.07 mmol, 840 lL) in DMF (20 mL)
was stirred at room temperature for 30 min. 9-Aminoacridine
hemihydrate (1.05 g, 4.22 mmol) was added dropwise to the acti-
vated ester. The reaction mixture was stirred for 24 h at 50 °C to al-
low the complete dissolution of the acridine. Almost all of the DMF
was removed under reduced pressure and the residue was added
dropwise to a stirred solution of 5% NaHCO3 (100 mL). The mixture
was allowed to stand for 24 h at room temperature. The resulting
precipitate was filtered and dried under vacuum and [2-(acridin-
9-ylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester was obtained
as an amorphous powder in 60% yield (2.52 mmol, 920 mg), after
chromatography in CH2Cl2/MeOH (95:5).
13C NMR (CDCl3, 63 MHz) d ppm: 26.69 (2 CH2), 31.73 (2 CH2),
50.6 (2 CH2), 99.1 (2 O–CH3), 116.1 (2 C Acr), 123.8 (2 CH Acr),
124.4 (2 CH Acr), 124.8 (2 CH Acr), 128.5 (2 CH Acr), 131.8 (2 CH
Acr), 148.4 (4 C Acr), 149.2 (2 C Acr), 149.5 (2 C Acr), 153.2 (2 C
Acr), 156.1 (2 C Acr).
MS (ES) m/z: 599.2 (MH+) (100%), 300.1 (M+2H+)/2 (26.4%).
Anal. Calcd for C34H32Cl2N4O2ꢁ2.5H2O: C, 63.35; H, 5.78; N, 8.69.
Found: C, 63.82; H, 5.72; N, 8.01. MM = 644.6.
1H NMR (CD3OD, 360 MHz) d ppm: 1.50 (s, 9H–3 CH3 Boc), 2.96
(m, 2H–CH2 CO bala), 3.55 (t, J = 6.5 Hz, 2H–CH2 NH bala), 7.64 (t,
J = 8.0 Hz, 2H–2 CH Acr), 7.88 (t, J = 8.0 Hz, 2H–2 CH Acr), 8.19 (d,
J = 8.0 Hz, 4H–4 CH Acr).
6.1.5. N1-Acridin-9-yl-hexane-1,6-diamine (6)
A solution of 9-chloroacridine (2.49 mmol, 0.531 g), NH-Boc-
hexylamine (2.81 mmol, 0.607 g) and DIEA (4.98 mmol, 822 lL)
was heated at 60–80 °C for 6 h. After evaporation under reduced
pressure and purification by MPLC with CH2Cl2/MeOH (2–10%
MeOH), the amino derivative was obtained in 66% yield
(1.77 mmol, 0.65 g).
This amino compound was then dissolved in TFA (20 mL) and
stirred overnight at room temperature. After evaporation under re-
duced pressure, the crude product was passed over an ion ex-
13C NMR (CD3OD, 50 MHz) d ppm: 29.0 (3 CH3 Boc), 37.5 (CH2),
38.2 (CH2), 80.5 (C Boc), 124.5 (CH Acr), 125.5 (CH Acr), 127.6 (CH
Acr), 129.5 (C Acr), 132.2 (CH Acr), 150.4 (2 C Acr), 158.6 (CO car-
bamate), 174.0 (CO amide).
MS (ES) m/z: 366.2 (MH+) (100%), 388.2 (MNa+) (45.4%).
6.1.7.2. 2-(Acridin-9-ylcarbamoyl)ethyl-ammonium chloride. [2-
(Acridin-9-ylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester
(2.52 mmol, 920 mg) was dissolved in TFA/CH2Cl2 (1:1) (10 mL).
Afterstirringovernightin thedark, thesolventwas evaporatedin va-
cuo. The TFA salt 8 was obtained quantitatively and used without
further purification.
change resin Dowex Clꢀ, which afforded compound
lyophilization in 60% yield (1.40 mmol, 0.548 g).
6 after
1H NMR (CD3OD, 360 MHz) d ppm: 1.53 (m,4H–2CH2), 1.72 (m,
2H–CH2), 2.02 (m, 2H–CH2), 2.95 (t, J = 7.5 Hz, 2H–CH2), 4.14 (t,
J = 7.4 Hz, 2H–CH2), 7.52 (t, J = 7.4 Hz, 2H–2CH Acr), 7.79 (d,
J = 8.1 Hz, 2H–2CH Acr), 7.90 (t, J = 7.4 Hz, 2H–CH Acr), 8.47 (d,
J = 8.4 Hz, 2H–2CH Acr).
6.1.7.3. {2-[2-(Acridin-9-ylcarbamoyl)-ethylcarbamoyl]-ethyl}-
carbamic acid tert-butyl ester. A mixture of Boc-b-AlaOH
(2.77 mmol, 525 mg) and CDI (2.77 mmol, 450 mg) in DMF
(10 mL) was stirred at room temperature for 30 min. The TFA salt
8 and DIEA (3.78 mmol, 625 lL) were dissolved in DMF (10 mL)
and added dropwise to the activated ester. The reaction mixture
was stirred overnight in the dark at room temperature. DMF was
removed under reduced pressure and the residue was dissolved
in acetone (10 mL) and added dropwise to a stirred solution of
5% NaHCO3 (100 mL). The mixture was allowed to stand for 24 h
at room temperature. The resulting precipitate was filtered and
13C NMR (CDOD3, 50.3 MHz) d ppm: 27.3 (CH2), 27.6 (CH2), 28.6
(CH2), 30.7 (CH2), 40.9 (CH2), 50.5 (CH2), 120.1 (2 C Acr), 124.0 (2
CH Acr), 125.7 (2 CH Acr), 128.9 (2 CH Acr), 132.7 (2 CH Acr),
145.5 (C Acr), 149.2 (2 C Acr).
MS (ES) m/z: 368, (100%) (MH+).
HRMS calcd for C19H23N3 (MH+): 294.1970, found: 294.1958.
Anal. Calcd for C19H23N3ꢁ2HClꢁ1.5H2Oꢁ0.25NaCl: C, 58.52; H,
6.72; N, 10.77. Found: C, 58.52; H, 6.85; N, 10.52. MM = 389.95.
UV–vis (H2O): kmax nm (
470), 420 (3 930), 442 (3 310).
e
molꢀ1 L cmꢀ1) = 276 (18 400), 340 (2

8038
L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
dried under vacuum. {2-[2-(acridin-9-ylcarbamoyl)-ethylcarba-
moyl]-ethyl}-carbamic acid tert-butyl ester was obtained as a pale
yellow powder in 78% yield (1.97 mmol, 860 mg).
conditions that consisted of 10% O2, 5% CO2, and 85% N2 at 37 °C
with a humidity of 95%.
1H NMR (CD3OD, 250 MHz) d ppm: 1.39 (s, 9H–3 CH3 Boc), 2.50
(t, J = 6.8 Hz, 2H–CH2 CO bala2), 2.96 (t, J = 6.8 Hz, 2H–CH2 CO
bala1), 3.35 (t, J = 6.8 Hz, 2H–CH2 NH bala2), 3.68 (t, J = 6.8 Hz,
2H–CH2 NH bala1), 7.59 (t, J = 7.5 Hz, 2H–2 CH Acr), 7.83 (t,
J = 7.5 Hz, 2H–2 CH Acr), 8.15 (t, J = 7.5 Hz, 4H–4 CH Acr).
13C NMR (CDCl3, 50 MHz) d ppm: 28.7 (3 CH3 Boc), 37.1 (CH2
bala), 37.2 (CH2 bala), 37.3 (CH2 bala), 37.4 (CH2 bala), 86.1 (C
Boc), 123.8 (2 CH Acr + 2 C Acr), 125.5 (2 CH Acr), 126.7 (2 CH
Acr), 129.0 (2 C Acr), 132.0 (2 CH Acr), 150.0 (2 C Acr), 164.8 (CO
carbamate), 173.4 (CO amide), 174.1 (CO amide).
6.2.2. Measurement of in vitro antimalarial activity
Solutions of drugs were prepared in RPMI 1640 medium and
distributed in triplicate into Falcon 96-well flat-bottomed plates
(Becton Dickinson, Franklin Lakes, NJ) to achieve concentrations
ranging from 0.006
For in vitro isotopic microtests, 25
200 L/well of the suspension of synchronous parasitised red
lM to 200 lM.
lL/well of compounds and
l
blood cells (final parasitemia, 0.5%; final hematocrit, 1.5%) were
distributed in 96-well plates. Parasite growth was assessed by add-
ing 1 l
Ci of [3H]hypoxanthine with a specific activity of 14.1 Ci/
MS (ES) m/z: 437.3 (MH+) (100%), 459.2 (MNa+) (99.6%).
mmol (Perkin–Elmer, Courtaboeuf, France) to each well. Plates
were incubated for 42 hours at 37 °C in an atmosphere of 10%
O2, 5% CO2, 85% N2, and an humidity of 95%. Immediately after
incubation the plates were frozen then thawed to lyse erythro-
cytes. The contents of each well were collected on standard filter
microplates (UnifilterTM GF/B, Perkin–Elmer, Meriden, USA) and
washed using a cell harvester (FilterMateTM Cell Harvester, Pack-
6.1.7.4. 2-[2-(Acridin-9-ylcarbamoyl)-ethylcarbamoyl]-ethyl-
ammonium chloride (8). The previous compound (1.97 mmol,
860 mg) was dissolved in TFA/CH2Cl2 (1:1) (10 mL). After stirring
overnight in the dark, the solvent was evaporated in vacuo. The so-
lid was dissolved in distilled water and passed over an ion ex-
change resin Dowex Clꢀ. The chloride salt 2-[2-(acridin-9-
ylcarbamoyl)-ethylcarbamoyl]-ethyl-ammonium chloride (8) was
obtained quantitatively, after lyophilization.
ard). Filter microplates were dried and 25 lL of scintillation cock-
tail (MicroscintTM O, Perkin–Elmer) was placed in each well.
Radioactivity incorporated by the parasites was measured using a
scintillation counter (Top CountTM, Perkin–Elmer).
1H NMR (CD3OD, 250 MHz) d ppm: 2.67 (t, J = 6.5 Hz, 2H–CH2
CO bala2), 3.12 (t, J = 6.5 Hz, 2H–CH2 CO bala1), 3.22 (t, J = 6.5 Hz,
2H–CH2 NH bala2), 3.71 (t, J = 6.5 Hz, 2H–CH2 CH2 NH bala1),
7.91 (t, J = 8.0 Hz, 2H–2 CH Acr), 8.28 (q, J = 8.0 Hz, 4H–4 CH Acr),
8.15 (d, J = 8.0 Hz, 2H–2 CH Acr).
The 50% inhibitory concentration (IC50), that is, the drug con-
centration corresponding to 50% of the uptake of [3H]hypoxanthine
by the parasites in drug-free control wells, was determined by non-
linear regression analysis of log-dose/response curves (RiasmartTM,
Packard, Meriden, USA). Data were analysed after logarithmic
transformation and expressed as the geometric mean IC50 and
95% confidence intervals (95% CI) were calculated (Stata9TM, Stata-
Corp LP, Texas, USA).
MS (ES) m/z : 337.2 (MH+) (55%) 359.1 (MNa+) (45%).
HRMS calcd for C19H20N4O2 (MH+): 337.1665, found: 337.1660.
Anal. Calcd for C19H23ClN4O2ꢁ0.75NaClꢁH2O: C, 52.50; H, 5.33; N,
12.89. Found: C, 52.41; H, 5.39; N, 12.72. MW: 434.70.
UV–vis (H2O): kmax nm (
480).
e
molꢀ1 L cmꢀ1) = 251 (97 600), 360 (9
6.2.3. Cell culture and cell proliferation assay
The human cell lines KB (month epidermoid carcinoma) was
obtained from ECACC (Salisbury, UK) and grown in D-MEM med-
ium supplemented with 10% fetal calf serum (Invitrogen), in the
presence of penicillin, streptomycin and fungizone in 75 cm2 flask
under 5% CO2. Cells were plated in 96-well tissue culture micro-
6.1.8. pKa determination
Spectrophotometric measurements were carried out to deter-
mine pKa values. Solutions of acridine derivatives were prepared
at a final concentration of 2.5
taining 0.15 M NaCl. Titration were conducted with NaOH solu-
tions (0.4 M or 0.1 M) containing 2.5 M of acridine to avoid
dilution effects and 0.15 M NaCl. Spectra were recorded between
200 and 500 nm after each addition leading to an increase of 0.5
pH unit (or 0.25 pH unit in the pKa zone). A sigmoid curve was then
obtained by plotting pH versus AkAcrH/AkAcr, where kAcrH and kAcr
are respectively the maximum wavelengths of the protonated and
the neutral species, and AkAcrH and AkAcr the absorbances corre-
sponding to these specific wavelengths. The pKa value was calcu-
lated using the following expression in KGraph 5.1 to fit the curve:
lM in hydrochloric acid 0.2 M con-
plates at a density of 650 cells/well in 200
24 h later with compounds dissolved in DMSO at concentrations
ranged from 0.5 nM to 10 M using a Biomek 3000 automate
lL medium and treated
l
l
(Beckman-Coulter). Controls received the same volume of DMSO
(1% final volume). After 72 h exposure MTS reagent (Promega)
was added and incubated for 3 h at 37 °C: the absorbance was
monitored at 490 nm and results expressed as the inhibition of cell
proliferation calculated as the ratio [(OD490 treated/OD490 con-
trol) ꢂ 100]. For IC50 determinations (50% inhibition of cell prolif-
eration) experiments were performed in separate duplicate.
AkAcrH 10ꢀpHAkAcrHðAcrHþÞ þ KaAkAcrHðAcrÞ
6.3. Interaction with hematin
¼
10ꢀpHAkAcrðAcrHþÞ þ KaAkAcrðAcrÞ
AkAcr
6.3.1. Reagents
Hemin chloride (H-5533), hematin (ferriprotoporphyrin IX
hydroxide) (H-3281), chloroquine diphosphate (C-6628) were ob-
tained from Sigma.
6.2. Biological evaluation
6.2.1. P. falciparum strains
Both CQ-susceptible (3D7) and CQ-resistant (W2, FCR3 and
Bre1) P. falciparum strains maintained continuously in culture were
used. All strains were synchronised twice with sorbitol before use.
Synchronous parasites were diluted with uninfected erythrocytes
(A-positive human blood) and completed RPMI 1640 medium
(Invitrogen, Paisley, United Kingdom), supplemented with 10% hu-
man serum Abcys S.A. (Paris, France) and buffered with 25 mM
HEPES and 25 mM NaHCO3 to achieve 0.5 parasitemia and 1.5
hematocrit. Parasites were grown under controlled atmospheric
6.3.2. Microassay for b-hematin formation: BHIA
The method to assay the ability of different compounds to inhi-
bit b-hematin formation was reported for the first time by Parapini
et al.,24 and is referred as BHIA (b-hematin inhibitory activity).
A total of 50
l
L of an 8 mM solution of hemin dissolved in
DMSO was introduced in eppendorfs (0.4
lmol/eppendorf); 50 lL
of different compounds in DMSO or DMSO/MeOH (1:1), in doses
ranging from 0 to 10 M equiv to hemin, was added to triplicate test
eppendorfs. In control eppendorfs, 50 lL of water was added

L. Guetzoyan et al. / Bioorg. Med. Chem. 17 (2009) 8032–8039
8039
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Eppendorfs were incubated at 37 °C for 18 h to allow complete
reaction and were then centrifuged 4000g for 15 min. The soluble
fraction of unprecipitated hematin was collected. The remaining
pellet was resuspended with 200 lL of DMSO to remove unreacted
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6.3.3. Drug/hematin interaction assay: determination of Kd and
stoichiometry
An aqueous DMSO (40%, v/v) solution of 10
lM hematin (pH
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at a speed of 0.5 nm/min. Spectrophotometric data could then be
analyzed by the standard equation36 1/
D
A = 1/
D
A
1
+ Kd/D
A1 ꢂ 1/
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[Drug]n, where
D
A = A ꢀ A0,
DA1 = A ꢀ A and A0, A and A are
1
1
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the absorbance of the initial, final and mixed species, respectively.
The linearity of the graph representing 1/ A as a function of 1/
D
[Drug]n was then assayed with n = 1, n = 2/3, n = 3/2. . . and Kd
and A could be determined graphically.
1
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We are grateful to R. Amalvict, E. Baret, and J. Mosnier (Institut
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assistance and availability in this work.
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Supplementary data
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